Regulating voltage-gated ion channels with nanobodies

In this work, Morgenstern and colleagues describe an approach involving functionalized nanobodies which decrease the activity of voltage-gated Ca2+ channels associated with β1 subunits and promote their removal from the surface membrane of neurons and muscle.

channels, this pharmacological approach does not afford precise, tissue-specific regulation of Ca 2+ entry. Gene ablation or siRNAmediated protein knockdown of β or other subunits approaches could circumvent these limitations, but the interpretation of these experiments is confounded when there are multiple auxiliary subunit isoforms expressed in a cell, some of which have partially overlapping functions.

Nanobodies target ion channels of specific composition with precision
Here, Morgenstern et al. 1 describe an elegant and highly effective strategy to decrease the functional impact of β 1 -associated Ca V channels using nanobodies.
Nanobodies are recombinant antigen binding fragments, and their small size and folding properties enhance their stability inside of live cells, where they can be utilized as "intrabodies" 10 . Morgenstern et al. 1 demonstrate how Ca V channels may be targeted with functionalized nanobodies, to precisely inhibit channels comprising of specific β subunit isoforms.
In their previous work, Morgenstern et al. 11 demonstrated a Ca V β-targeted nanobody (nb.F3) inhibits Ca V 1/2 channels by initiating their redistribution into endosomes. This nanobody-delivered ubiquitination machinery (Ca V -aβlator) functions as an effective inhibitor of Ca V channels. In this present work, Morgenstern et al. 1 reveal a refined inhibitor specifically targeted to β 1 -associated Ca V channels (Chisel-1).
Briefly, the authors identified a nanobody (nb.E8) which selectively binds the Ca V β 1 SH3 domain and inhibits Ca V β 1 -associated voltage-gated Ca V channels by decreasing open probability and increasing their rate of channel inactivation. Interestingly, nb.E8 also decreases channel activity by reducing channel surface density.
Functionalizing nb.E8 with the Nedd4L HECT domain yielded Chisel-1, which eliminated current through Ca V β 1 -reconstituted Ca V 1/ Ca V 2 and native Ca V 1.1 channels in skeletal muscle. Chisel-1 also decreased depolarization-induced Ca 2+ entry and excitationtranscription coupling in hippocampal neurons. Notably, Chisel-1 was ineffective against Ca V β 2 -associated Ca V 1.2 channels in cardiomyocytes, underscoring its specificity. In a therapeutic setting, genetically-encoded inhibitors like Ca V -aβlator and Chisel-1 could be selectively expressed within cells of interest, potentially bypassing the off-target effects produced by many traditional Ca V inhibitors.

Nanobodies could reveal important aspects of ion channel organization and function
The findings by Morgenstern et al. 1 raise multiple intriguing questions. For example, how does binding of the nb.E8 nanobody to Ca V β 1 , independent of ubiquitin ligase conjugation, act to reduce the membrane surface density of Ca V 2.2 channels? Does the reduction in channel activity in the presence of nb.E8 prime the channel for nature communications (2022) 13:7557 | endocytosis? Also, Ca V channels form clusters in the surface membrane of neurons and muscle 12,13 . Recent studies indicate that ion channels involved in cooperative signaling cascades co-cluster. This raises the question of whether Chisel-1-bound channels are removed individually within a cluster or if the binding of a subset of channels destines the entire cluster for removal? The latter mechanism would suggest an amplification mechanism which could impact on clustered channels. Future studies should investigate these questions.